Signe Danielsen

Improved chitosan-mediated gene delivery based on easily dissociated chitosan polyplexes of highly defined chitosan oligomers

Nonviral gene delivery systems based on conventional high-molecular-weight chitosans are efficient after lung administration in vivo, but have poor physical properties such as aggregated shapes, low solubility at neutral pH, high viscosity at concentrations used for in vivo delivery and a slow dissociation and release of plasmid DNA, resulting in a slow onset of action. We therefore developed highly effective nonviral gene delivery systems with improved physical properties from a series of chitosan oligomers, ranging in molecular weight from 1.2 to 10 kDa. First, we established structure–property relationships with regard to polyplex formation and in vivo efficiency after lung administration to mice. In a second step, we isolated chitosan oligomers from a preferred oligomer fraction to obtain fractions, ranging from 10 to 50-mers, of more homogeneous size distributions with polydispersities ranging from 1.01 to 1.09. Polyplexes based on chitosan oligomers dissociated more easily than those of a high-molecular-weight ultrapure chitosan (UPC, approximately a 1000-mer), and released pDNA in the presence of anionic heparin. The more easily dissociated polyplexes mediated a faster onset of action and gave a higher gene expression both in 293 cells in vitro and after lung administration in vivo as compared to the more stable UPC polyplexes. Already 24 h after intratracheal administration, a 120- to 260-fold higher luciferase gene expression was observed compared to UPC in the mouse lung in vivo. The gene expression in the lung was comparable to that of PEI (respective AUCs of 2756±710 and 3320±871 pg luciferase × days/mg of total lung protein). In conclusion, a major improvement of chitosan-mediated nonviral gene delivery to the lung was obtained by using polyplexes of well-defined chitosan oligomers. Polyplexes of oligomer fractions also had superior physicochemical properties to commonly used high-molecular-weight UPC.

Evaluation of the use of Sr2+ in alginate immobilization of cells.

Abstract. In recent years the method of immobilization of living cells in Ca-alginate beads has gained a wide range of applications. In all cases high chemical stability of the immobilization material and mild conditions for the cells are prerequisites. However, in long-term experiments that may last for several days Ca-alginate may dissolve due to an exchange of Ca2+ with Na+, forming fluid Na-alginate. As well as Ca-alginate, the more chemically stable Sr-alginate and Ba-alginate are materials that have been used for the immobilization of living cells. In this study, the effects of Ca2+, Sr2+ and Ba2+ on growth, viability and intracellular free calcium concentration in a human leukemic T cell line (Jurkat) were investigated. The findings in this study, and the fact that Sr-alginate has a considerably higher chemical stability than Ca-alginate, led to the conclusion that Sr-alginate is a more suitable material for use in the entrapment of living cells in long-term studies.

Adaptive brachytherapy of cervical cancer, comparison of conventional point A and CT based individual treatment planning.

Abstract Background. Locally advanced cervical cancer is commonly treated with external radiation therapy combined with local brachytherapy. The brachytherapy is traditionally given based on standard dose planning with prescription of dose to point A. Dosimetric aspects when changing from former standard treatment to individualized treatment plans based on computed tomography (CT) images are here investigated. Material and methods. Brachytherapy data from 19 patients with a total of 72 individual treatment fractions were retrospectively reviewed. Standard library plans were analyzed with respect to doses to organs at risk (OARs), and the result was compared to corresponding delivered individualized plans. The theoretical potential of further optimization based on prescription to target volumes was investigated. The treatments were performed with a Fletcher applicator. Results. For standard treatment planning, the tolerance dose limits were exceeded in the bladder, rectum and sigmoid in 26%, 4% and 15% of the plans, respectively. This was observed most often for the smallest target volumes. The individualized planning of the delivered treatment gave the possibility of controlling the dose to critical organs to below certain limits. The dose was still prescribed to point A. An increase in target dose coverage was achieved when additional individual optimization was performed, while still keeping the dose to the OARs below predefined limits. Relatively low average target coverage, especially for the largest volumes was however seen. Conclusion. The individualized delivered treatment plans ensured that doses to OARs were within acceptable limits. This was not the case in 42% of the corresponding standard plans. Further optimized treatment plans were found to give an overall better dose coverage. In lack of MR capacity, it may be favorable to use CT for planning due to possible protection of OARs. The CT based target volumes were, however, not equivalent to the volumes described in magnetic resonance imaging (MRI) based recommendations. Prescription and assessment of dose, when introducing such target volumes, should be evaluated and preferably compared to well known treatment regimens.

Dosimetric evaluation of manually and inversely optimized treatment planning for high dose rate brachytherapy of cervical cancer.

Abstract Background. To compare five inverse treatment planning methods with the conventional manually optimized planning approach for brachytherapy of cervical cancer with respect to dosimetric parameters. Material and methods. Eighteen cervical cancer patients treated with magnetic resonance imaging (MRI)-guided high dose rate (HDR) brachytherapy were included in this study. Six plans were created for each of the 4 HDR brachytherapy fractions for each patient: 1 manually optimized and 5 inversely planned. Three of these were based on inverse planning simulated annealing (IPSA) with and without extra constraints on maximum doses of the target volume, and different constraints on doses to the organs at risk (OARs). In addition there were two plans based on dose to target surface points. The resulting dose-volume histograms were analyzed and compared from the dosimetric point of view by quantifying specific dosimetric parameters, such as clinical target volume (CTV) D90, CTV D100, conformal index (COIN), and D2cm3 for rectum, bladder and the sigmoid colon. Results. Manual optimization led to a mean target coverage of 78.3% compared to 87.5%, 91.7% and 82.5% with the three IPSA approaches (p < 0.001). Similar COIN values for manual and inverse optimization were found. The manual optimization led to better results with respect to the dose to the OARs expressed as D2cm3. Overall, the best results were obtained with manual optimization and IPSA plans with volumetric constraints including maximum doses to the target volume. Conclusions. Dosimetric evaluation of manual and inverse optimization approaches is indicating the potential of IPSA for brachytherapy of cervical cancer. IPSA with constraints of maximum doses to the target volume is closer related to manual optimization than plans with constraints only to minimum dose to the target volume and maximum doses to OARs. IPSA plans with proper constraints performed better than those based on dose to target surface points and manually optimized plans.

Relative biological effectiveness of photon energies used in brachytherapy and intraoperative radiotherapy techniques for two breast cancer cell lines.

Abstract Background. Partial breast irradiation (IORT or brachytherapy) differ from external radiation of whole breast in terms of irradiated volumes, fractionation, radiation energy and dose rate; all factors influencing the treatment outcome in a complex manner. Theoretically obtained RBE values comparing effects of radiation used in IORT and external therapy are published, but experimental studies are required to confirm these data. The aim of this study is to establish such RBE values for two breast cancer cell lines. Materials and methods. Colony formation of breast cancer cell lines (MCF-7 and T-47D) were studied after photon irradiation with qualities and dose rates used in IORT, brachytherapy and external radiation. RBE values from survival data were used to compare effects. Results. Increasing the photon energy (dose rate 0.2 Gy/min) from 50 kV (Intrabeam) to 380 keV (192Ir source) and 6 MV (linear accelerator) yielded an increase in the cell survival, whereas increasing the dose rate to 6 Gy/min had minor effect. Average RBE values for 50 kV with 6 MV as reference radiation varied from about 1.4 (for doses < 5 Gy) to > 1.9 (for doses < 0.02 Gy) for MCF-7 cells and from about 1.4 to > 3.1 for T-47D cells for the same dose levels. Corresponding RBE values for 380 keV radiation were about 1.4 for MCF-7 cells and 1.3–2.3 for T-47D cells. Conclusion. RBE data for breast cancer cells exposed to radiation used in IORT, brachytherapy or external radiation differ among the cell lines tested. The values are in agreement with published theoretical and experimental work.